PROJECT RESEARCH WORK

ON

ISOLATION AND IDENTIFICATION OF HEAVY METAL-TOLERANT

FUNGI

BY:

HND/22/SLT/FT/

SUBMITTED TO:
THE DEPARTMENT OF SCIENCE LABORATORY
TECHNOLOGY, INSTITUTE OF APPLIED SCIENCES, (IAS)
KWARA STATE POLYTECHNIC, ILORIN.
IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE
HIGHER NATIONAL DIPLOMA (HND) SCIENCE LABORATORY
TECHNOLOGY (MICROBIOLOGY OPTION)

2023/2024 SESSION
 ABSTRACT

The rapid growth of electronic waste (e-waste) poses significant environmental and health risks

due to the presence of toxic metals. This aim of this study analyzed the metal composition of e-

waste, specifically printed circuit boards (PCBs), and identify fungi tolerance to these metals.

AAS analysis was used to analyze metal composition of the PCBs and fungi were cultured on

liquid Luria bertani media using standard methods. The results showed a diverse metal

composition, with iron, zinc, nickel, and copper, with Iron (5.786ppm) having the highest

composition while cupper (1.442ppm) had the lowest. The fungi tolerant to high metals in E-

waste includes, Aspergillus spp., Fusarium spp., Saccharomyces cerevisiae., making them

potential candidates for bioremediation and bioleaching applications.

 CHAPTER 1

INTRODUCTION

1.1. Background

Metal contamination is a significant and pervasive environmental issue that has gained

increasing attention in recent decades. This problem has been exacerbated by various human

activities, such as mining, industrial processes, agricultural practices, and improper waste

disposal (Sharma and Dubey, 2005). The primary sources of metal contamination are diverse and

wide-ranging. Mining and smelting operations, where metals are extracted and processed, can

release large quantities of heavy metals, such as lead (Pb), cadmium (Cd), chromium (Cr),

copper (Cu), and zinc (Zn), into the surrounding environment (Nagajyoti et al., 2010). Industrial

activities, including electroplating and the manufacturing of batteries, paints, and other metal-

containing products, can also contribute to metal pollution through the discharge of contaminated

effluents. Agricultural practices, such as the application of metal-based pesticides and fertilizers,

can lead to the accumulation of heavy metals in soil and water bodies. Furthermore, the

combustion of fossil fuels and the improper disposal of electronic waste (e-waste) can release

various heavy metals into the atmosphere, eventually depositing onto land and water

surfaces.The environmental impact of metal contamination is profound and far-reaching. Heavy

metals are non-biodegradable, meaning they do not break down over time, and they can persist in

the environment for extended periods. These metals can accumulate in soil, sediments, and living

organisms, leading to the disruption of ecosystems and the potential for biomagnification

through the food chain (Alloway, 2013). As heavy metals move up the food chain, their

concentrations can increase, posing a serious threat to the health of higher-level organisms,
including humans. Exposure to high levels of heavy metals can have devastating effects on

human health, leading to a range of neurological disorders, cancer, organ dysfunction, and other

severe health issues. Additionally, the presence of these contaminants in the environment can

have detrimental impacts on plant and animal life, affecting their growth, reproduction, and

overall ecological balance given the persistent and harmful nature of metal contamination, the

development of effective remediation strategies has become a pressing need. Conventional

methods, such as physical and chemical treatments, can be costly and may generate secondary

waste streams. In this context, bioremediation strategies, which utilize the capabilities of

microorganisms, including fungi, have emerged as a promising and more environmentally

friendly approach (Gadd, 2010). These biological methods offer the potential for cost-effective

and sustainable solutions to tackle the pressing issue of metal contamination.

1.2. Importance of metal-tolerant fungi in bioremediation and resource recovery

The increasing prevalence of metal contamination in the environment has spurred the search for

effective and sustainable remediation strategies. In this context, the use of metal-tolerant fungi

has emerged as a promising approach for addressing the challenges posed by heavy metal

pollution (Gadd, 2010).The application of metal-tolerant fungi in bioremediation has several

advantages. These microorganisms can be employed in ex situ or in situ remediation approaches,

offering flexibility in their implementation. Furthermore, the use of fungi is often more cost-

effective and environmentally friendly compared to traditional physicochemical treatment

methods (Javanbakht et al., 2014).In addition to their bioremediation capabilities, metal-tolerant

fungi can also play a role in resource recovery. The ability of these fungi to accumulate and

concentrate heavy metals within their biomass can be exploited for the recovery and recycling of

valuable metals from contaminated environments (Gadd, 2010). This not only addresses the issue
 of metal pollution but also contributes to the circular economy by recovering and repurposing

scarce and valuable resources. Numerous studies have demonstrated the potential of metal-

tolerant fungi in various applications, such as the removal of heavy metals from industrial

effluents, the remediation of contaminated soils, and the recovery of precious metals from

electronic waste (PCBs) (Javanbakht et al., 2014; Gadd, 2010). As the demand for sustainable

and environmentally friendly solutions continues to grow, the role of metal-tolerant fungi in

bioremediation and resource recovery is likely to become increasingly important.

1.3. Aim and Objectives of the study

Aim

The aim of this project is to isolate and identify metal-tolerant fungi from metal contaminated

soil samples and testing their mental tolerance ability in E-waste.

Objectives

The main objectives of the project on the isolation and identification of metal-tolerant fungi are:

i. To isolate bacteria from diverse environmental samples known to be contaminated with

heavy metals.

ii. To characterize the isolated bacteria morphologically, biochemically to identify their

species.

iii. To screen the isolated fungi for their mental tolerance ability in E-waste.
 CHAPTER 2

LITERATURE REVIEW

2.1. Overview of metal-tolerant fungi and their mechanisms of tolerance

In the ever-evolving tapestry of life, a remarkable group of organisms stands out as nature's

unsung heroes the metal-tolerant fungi. These resilient microbes, armed with an array of

ingenious strategies, have become the alchemists of the microbial world, transforming the

seemingly indestructible heavy metals into manageable forms (Gadd, 2010).Imagine a rugged

landscape, scarred by the relentless march of industry, where the very soil and water have been

tainted by the toxic fingerprints of heavy metals. It is in these challenging environments that the

metal-tolerant fungi thrive, defying the odds and thriving where others would succumb

(Javanbakht et al., 2014).At the heart of their remarkable resilience lies a suite of specialized

mechanisms that allow them to cope with the presence of these heavy hitters. Through the

process of biosorption, these fungi can act as living sponges, sequestering and accumulating

heavy metals on their cell walls and surfaces (Gadd, 1993). This physical adsorption not only

removes the contaminants from the surrounding environment but also concentrates them within

the fungal biomass. But the fungi's alchemical prowess doesn't stop there. Many species possess

the ability to actively transport and store heavy metals within their cells, a process known as

bioaccumulation. By compartmentalizing these toxic elements, they effectively remove them

from the system, reducing their bioavailability and the potential for harm (Gadd, 2010).Perhaps

the most remarkable feat of the metal-tolerant fungi is their capacity for biotransformation the

ability to chemically modify and detoxify heavy metals through their enzymatic and metabolic

machinery. These fungi can quite literally transform the very nature of the heavy metals,
rendering them less harmful and more easily managed (Gadd, 2010). It is this unique blend of

biosorption, bioaccumulation, and biotransformation that has made the metal-tolerant fungi the

darlings of the bioremediation world. Their ability to clean up the messes left behind by

industrial activities and mining operations has made them invaluable allies in the fight against

environmental degradation (Javanbakht et al., 2014). But the story of the metal-tolerant fungi

doesn't end there. These alchemists of the microbial world have also captured the attention of

those seeking to recover and recycle valuable metals from waste streams, such as electronic

waste (PCBs) (Gadd, 2010). By harnessing the fungi's capacity to concentrate and accumulate

precious metals, we can not only address the issue of pollution but also contribute to the circular

economy, ensuring that these scarce resources are reclaimed and repurposed. As the world

grapples with the growing challenge of heavy metal contamination, the metal-tolerant fungi

stand as beacons of hope, reminding us that nature's solutions are often the most elegant and

sustainable. These unsung heroes, with their extraordinary abilities, are poised to play a crucial

role in shaping a cleaner, greener future for all.

As the world grapples with the ever-growing challenge of heavy metal pollution, researchers

have turned their attention to the remarkable capabilities of metal-tolerant fungi. These resilient

microorganisms, armed with unique adaptations, have become the focus of intense scientific

exploration, as researchers seek to uncover their potential for bioremediation and resource

recovery.In the ongoing quest to harness the power of metal-tolerant fungi, a wealth of research

has been dedicated to the isolation and identification of these remarkable species. Through

meticulous sampling and isolation techniques, scientists have scoured diverse environments,

from contaminated soils to industrial waste streams, in search of these hidden gems of the

microbial world.One such pioneering study was conducted by Gadd and Griffiths (1978), who
investigated the presence of metal-tolerant fungi in a variety of metal-polluted habitats. The

researchers employed a range of culturing methods and selective media to isolate and identify a

diverse array of fungal species capable of thriving in the presence of heavy metals, such as

copper, zinc, and lead. Their findings revealed a remarkable diversity of metal-tolerant fungi,

including species from the genera Aureobasidium, Penicillium, and Trichoderma, among others.

Building upon this foundational work, numerous subsequent studies have expanded the catalogue

of metal-tolerant fungi. Javanbakht et al. (2014) explored the potential of fungi isolated from

metal-contaminated soils, identifying species from the genera Aspergillus, Mucor, and Rhizopus

as potent metal accumulators. Similarly, Baldrian et al. (2000) investigated the metal-tolerant

capabilities of wood-decay fungi, uncovering the remarkable adaptations of Pleurotus ostreatus

and Hypholoma fasciculare in the presence of copper and cadmium.The isolation and

identification of these metal-tolerant fungi have not only broadened our understanding of

microbial diversity but have also opened up new avenues for practical applications. By carefully

characterizing the physiological and genetic mechanisms that underlie metal tolerance,

researchers have paved the way for the development of innovative bioremediation and resource

recovery strategies. As the field of metal-tolerant fungal research continues to evolve, the

identification of novel species and the deepening of our knowledge about their unique

adaptations will undoubtedly lead to groundbreaking advancements. These unsung heroes of the

microbial world hold the potential to revolutionize the way we approach environmental cleanup

and resource management, ushering in a future where heavy metal pollution is a problem of the

past.
 CHAPTER 3

MATERIALS AND METHODS

3.1 Materials

Media (such as Saboraud Dextrose Agar), 70% Ethanol, Distilled water, Measuring tape,

Aluminum foil, Conical flask, Beaker, Test tubes, Marking pencil, Petri dish, Glass slide, Cover

slip, Syringes and Needles, Weighing balance, Refrigerator, Incubator, Microscope, Autoclave,

Spatula, Cotton wool, Stirring rod, Bunsen burner, Inoculating loop, Hand gloves, Face masks,

Measuring cylinder, Paper tape, Hot plate, Lactophenol cotton blue, AAS Machine and incubator

shaker.

3.2 Sample Collection

The samples for this study were collected from diverse environmental sources known to be

contaminated with heavy metals. These sources included:

E-waste

Electronic waste (e-waste) samples were collected from Challenge Ilorin, Kwara State, and

informal recycling site and area where electronic devices were being dismantled or recycled.

These site included scrapyards, electronic repair shops, and open dumping grounds where e-

waste accumulates.Personal protective equipment (PPE), including gloves and masks, was worn

to minimize exposure to hazardous materials.E-waste samples were collected by manually

selecting representative pieces of electronic devices, such as circuit boards, batteries, and

wires.Care was taken to collect samples from various types of electronic devices and components

to capture the diversity of metals present.Collected e-waste samples were placed in labeled
plastic bags or containers, ensuring that different types of waste were kept separate to avoid

mixing.

Soil Sample

Soil samples were collected from various locations including Sango Kulende Market, Ipata Oloje

spare part market within the market area, focusing on areas where activities involving the

handling and disposal of motor spare parts were prominent, sampling points were selected

randomly to ensure representative coverage of the site.Prior to collection, the sampling

equipment (Hand trowel) was cleaned and sterilized to prevent cross-contamination.Soil samples

were collected using a soil auger or shovel, and care was taken to collect samples from the

surface to a depth of at least 15-20 cm.Multiple subsamples were collected from different

locations within each sampling site and combined to obtain a representative composite

sample.The collected soil samples were then transferred into sterile, labeled containers and

sealed to prevent contamination during transport.

3.3 Preparation of E-Waste Materials

Source and Preparation of the Waste Materials

The PCBs was cleaned with distilled water, manually cut, crushed using a sheared cutters, and

sieved to particles smaller than 500 μm (micrometers) and grind with Electrical Grinder. And it

is necessary to first remove as much of the chemical coating as possible. PCBs powder was

transferred into a 10-M NaOH solution and left undisturbed for 48 h. After 48hrs PCBs was

taken out and washed under running tap water to remove the solder. The treated PCBs will be

kept in a tray dryer for 40 min and finally sieved for fine powder with a 120-μm pore size for

analysis and screen for bioleaching.

Fig 1: Powder and Granulated E- waste

Analysis of the E-wastes

The E-waste will be processed according to the protocol of Khatri (2018), and the particle size

will be reduced to have an approximate size of ≤ 200 to 250 μm, which will be used for this

study. 1g of finely powdered E-waste will be taken in a 100 mL Erlenmeyer flask and digested

using aqua regia, which can dissolve metals. Aqua regia is made up of Conc. HCl and HNO3 in

the ratio 3:1 (Sheng & Etsell 2007). The solution will be allowed to stand for 48 hours and then

will be centrifuged at 5000 rpm for 5 min. The aforementioned digested solution will be

analyzed by Atomic Absorption Spectrophotometry (AAS).

Fig 2:

Fig 3: PCBs powder in Aqua regia

3.4 Media Preparation

Saboraud Dextrose Agar (SDA) and Lauria Bertan (LB) broth was prepared according to

manufacturer specificationand then autoclave at 121 degree celcius for 15 minutes and some

biochemical reagents.

1. Saboraud Dextrose Agar (SDA) for isolation of fungi

2. Lauria Bertan (LB) broth

1. Saboraud Dextrose Agar (SDA) for isolation of fungi

Fifteen (15) grams of SDA powder was weighed using a weighing balance

in a sterile aluminium foil paper. The weighed media was poured into a

sterile conical flask 500ml containing 250ml of sterile distilled water. The

mouth of the flask was plugged with cotton wool and covered with

aluminium foil. The mixture was boiled for 15 minutes for homogeneity. It

was then autoclaved at 121°C for 15 minutes.

2. Lauria Bertan (LB) broth

Five (5g) grams of Pepton powder, 2.5 grams of Yeast Extract and 5grams Sodium Chloride

was weighed using balance into a sterile aluminium foil paper. Then weighed media was

poured into a sterile conical flask containing 500ml of sterile distilled water. The mouth was

covered with aluminium foil and cotton wool. And it was subjected to boiling for 15mins for

homogeneity. It was then autoclave at 121°C for enrichment of fungi.

3.4.1 Fungi Screening and Isolation

For microbial isolation, the soil sample collected from the Metal Contaminated sites, were

enriched in Luria broth (LB) medium (100 mL in 250 mL Erlenmeyer flask) containing sterilized

WPCBs. One gram (1g) of the representative soil sample will be inoculated in LB medium

supplemented with 1% pulp density of WPCBs (particle size ≤ 150 μm). The experimental flasks

will be incubated at 30°C in an incubator shaker at 150 rpm for 7 days. After 7days, 5 mL of

inoculum was transferred to the 2nd flask containing fresh medium supplemented with 2.5%

WPCBs followed by incubation under the same set of conditions for the next 7 days. The

sequential enrichment was continued for a period of 21 days at increasing concentrations of

WPCBs, upto 5%. After 21 days, samples from the flask containing 5% pulp density were

serially diluted and spread plated on Saboraud Dextrose Agar (SDA) plates. The plates were

incubated at 30°C for 2–4 days and observed for the appearance of colonies after every 24h.

Morphologically distinct colonies were selected and repeatedly sub-cultured to ensure the purity

of the bacterial isolates (Kumar et al. 2015). After 21 days, the content of all the flasks were

filtered and centrifuged at 7000 rpm for 10 min to remove the bacterial cells and residual

WPCBs from the medium. The supernatant obtained were further filtered through a whaltman

filter paper to ensure a particle-free suspension. The dissolution of metal ions will be analyzed

using AAS.
Fig 4: Filtration of medium incubated

3.4.2 Serial Dilution:

Serial dilution (101 to 107) was performed by transferring 1ml of broth from the enrichment

culture into 9ml of normal saline. The procedure was repeated to obtain the dilution of 107.

3.4.3 Isolation of Fungi:

Saboraud Dextrose Agar (SDA) plates were prepared according to standard protocols and

sterilized by autoclaving, Aliquots of the diluted samples were spread onto the surface of nutrient

agar plates using sterile spreaders. Multiple dilutions were plated to ensure that fungi colonies
 would be sufficiently separated for individual isolation. The inoculated nutrient agar plates were

then incubated at 37°C for 48 hours. After incubation, fungi colonies that appeared

morphologically distinct were selected for further analysis; Individual colonies were picked

using a sterile inoculation loop and streaked onto fresh saboraud dextrose agar plates for

purification. This process of colony isolation and subculture was repeated until pure fungi

cultures were obtained.

3.5 Identification of Fungi:

3.5.1 Morphological Characterization: Morphological characterization of fungi colonies

involves examining their shape, size, color, texture, and other physical characteristics.

i. Colony Morphology on Agar Plates

The overall appearance of bacterial colonies on agar plates is assessed. Characteristics such

as colony shape (e.g., round, irregular, filamentous), margin (e.g., entire, undulate, lobate),

elevation (e.g., flat, raised, convex), and surface texture (e.g., smooth, rough, mucoid) are

noted.

ii. Lactophenol procedure

The Lacto-phenol cotton blue staining procedure modified by (Morgan, 2007) was used in

the microscopic identification of fungi contaminants. The reagents used were Lacto-phenol

cotton blue reagent, a clean microscope slide was labeled with the code for the unknown

organism using a marking pencil. One to two drop of lacto-phenol cotton blue solution was

flooded on a slide and then, a small amount of unknown fungal colony culture was applied
 on the slide using an inoculating loop. Cover-slip was gently placed over the stained fungal

sample for a minutes before it was viewed under a microscope. The structure, such as

hyphae and spores were recorded (Morgan, 2007).

iii. Cellular Morphology: Bacterial cells are examined under a compound light microscope to

determine their cellular morphology, including shape (e.g., cocci, bacilli, spirilla),

arrangement (e.g., pairs, chains, clusters), and size.

 CHAPTER 4

RESULTS

AAS ANALYSIS OF UNTREATED AND TREATED PCBs IN NaOH

TABLE 1: PCBs BEFORE AFTER TREATED WITH NaOH

S/N METALS UNTREATED TREATED

CONTENTS POWDER GRANULATED

1 Iron (Fe) 5.786 1.616 1.429

2 Cupper (Cu) 1.442 1.481 1.511

3 Nickel (Ni) 1.549 2.063 2.539

4 Zinc (Zn) 2.775 1.673 2.159

4.1 ISOLATION AND SCREENING OF FUNGI

TABLE 2: MORPHOLOGICAL IDENTIFICATION OF ISOLATE

S/N SAMPLE NAME AND ORGANISMS DETECTED

LOCATION

Fig 5A E-waste Fusarium species

Fig 5B E-waste Aspergillus fumigatus

Fig 6 Sango Aspergillus flavus

Fig 7 Cement Aspergillus niger

Fig 8 Ipata Yeast cell (Saccharomyces

cerevisiae)
 Fig5A: Fusarium species
Fig 5B: Aspergillus fumigatus

Fig 6: Aspergillus flavus

Fig 7: Aspergillus niger

Fig8: Yeast cell (Saccharomyces cerevisiae)

4.3 ANALYTICAL OF ATOMIC SPECTROSCOPY

TABLE 3: AAS OF 1ST WEEK (7 DAYS) ISOLATE

S/N SAMPLE NAME Iron Cupper Nickel Zinc

(Fe) (Cu) (Ni) (Zn)

1 SS + PEW 1.006 1.404 3.148 1.325

2 IS + PEW 3.055 2.542 0.758 1.812

3 IS + GEW 1.712 1.465 2.39 1.85

4 SS + GEW 1.790 1.462 2.357 1.848

5 CS + GEW 1.488 1.451 0.994 0.272

SS + PEW = Sango soil & Powder E-Waste

IS + PEW = Ipata soil & Powder E-Waste

IS + GEW = Ipata soil & Granulated E-Waste

SS + GEW = Sango soil & Granulated E-Waste

CS + GEW = Cement soil & Granulated E-Waste

TABLE 4: AAS OF 2ND WEEK (14 DAYS) ISOLATE

S/N SAMPLE NAME Iron Cupper Nickel Zinc

(Fe) (Cu) (Ni) (Zn)

1 SS + PEW 1.225 0.927 3.403 1.616

2 IS + PEW 2.347 8.489 3.302 1.952

3 IS + GEW 1.614 7.941 4.525 1.987

4 SS + GEW 1.541 7.871 4.528 1.987

5 CS + GEW 1.749 7.556 2.993 1.374

TABLE 5: AAS OF 3RD WEEK (21 DAYS) ISOLATE

S/N SAMPLE NAME Iron Cupper Nickel Zinc

(Fe) (Cu) (Ni) (Zn)

1 SS + PEW 1.443 0.449 3.658 1.906

2 IS + PEW 1.638 14.429 5.846 2.092

3 IS + GEW 1.516 14.416 6.659 2.123

4 SS + GEW 1.292 14.279 6.699 2.126

5 CS + GEW 1.505 13.661 4.992 2.476

CHAPTER 5
 DISCUSSION AND CONCLUSION

5.1 DISCUSSION

Before Treatment: Iron (Fe) levels were notably high, with concentrations ranging from 5.7 to

86. Copper (Cu) levels were relatively low, measuring 1.442. After Treatment with NaOH:

Nickel (Ni) levels increased significantly, with concentrations of 2.063 in powdered form and

2.539 in granulated form. Copper (Cu) levels remained low, with slight increases to 1.481 in

powdered form and 1.511 in granulated form.

The fungi isolated in this study, Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, and

Saccharomyces cerevisiae, are known for their resilience and metal tolerance. These fungi were

cultured from treated PCBs samples on media containing different metal concentrations,

demonstrating their ability to thrive in metal-rich environments. Known for its high tolerance to

heavy metals, Aspergillus fumigatus by Doku and Belford (2015), demonstrated significant

bioleaching capabilities, particularly for metals such as Fe and Ni. Its metabolic processes enable

it to solubilize these metals, making them more accessible for recovery. Aspergillus flavus

showed moderate bioleaching abilities. It was effective in extracting metals like Cu and Zn from

e-waste. This fungus is also known for producing organic acids, which can facilitate metal

solubilization. One of the most effective fungi in this study, Aspergillus niger exhibited strong

bioleaching potential for a range of metals, including Fe, Cu, Ni, and Zn. Its production of citric

acid is particularly effective in chelating and mobilizing metals from solid waste matrices. While

primarily known as a model organism in fermentation, Saccharomyces cerevisiae also

demonstrated metal tolerance and bioleaching potential Salah and Anees (2015), it was effective
in solubilizing Cu and Ni, although its performance was generally lower compared to the

Aspergillus species. The results from the second week indicate a substantial increase in copper

levels across most samples, particularly for Ipata soil and granulated e-waste, which saw copper

concentrations rise to 8.489 and 7.941, respectively. Nickel levels also increased across all

samples, reflecting the dynamic changes in metal bioavailability over time. In Third week, the

results show a further increase in copper and nickel concentrations, particularly for Ipata and

Sango soil samples combined with Electronic waste (PCBs).

5.2 CONCLUSION
Fungi capable of tolerating heavy metals such as Cupper, Iron, Nickel, and Zinc from Electronic

waste (PCBs). The identified species, including Fusarium species, Aspergillus fumigatus,

Aspergillus niger and Yeast cell such as Saccharomyces cerevisiae. These fungi hold significant

potential for bioleaching applications, offering eco-friendly and cost-effective solutions for

mining of metals. Future research should focus on genetic pathways and the practical

implementation of these fungi in diverse environmental settings, enhancing their role in

sustainable environmental management.

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